Chitin is a linear insoluble polymer of β(1-4)-linked N-acetylglucosamine, which is a common consistent of fungal cell walls, shells of crustaceans, and exoskeletons of insects. It is the second most abundant polymer in nature and each year more than one billion tons of chitin is produced in the biosphere, mainly by insects, fungi, crustaceans, and other marine organisms.
In nature, two major types of chitin occur that are the characterized by an anti-parallel (α-chitin) or a parallel (β-chitin) arrangement of the polymer chains.
Despite its resilience, insolubility, and abundant production, chitin does not accumulate in most ecosystems, suggesting that nature has developed efficient processes for chitin degradation. Chitin is degraded for example by chitinases. Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi.
Chitinases have been cloned from various species of microorganisms and have been categorised into two distinct families, designate family 18 and family 19 of the glycoside hydrolases, based on sequence similarities (Henrissat and Bairoch, Biochem, J. 293: 781-788 (1993)).
In addition to being important for biomass turnover, chitin degradation is essential in a variety of biological processes. For example, plants are known to produce chitinases in response to attack by chitin-containing fungi, whereas some non-pathogenic fungi such as Trichoderma species are considered as “biocontrol” agents because of their ability to inhibit other, chitin-containing fungi. While chitin-containing organisms may be inhibited by exogenous chitinolytic activity, they do need some endogenous chitin turnover as part of morphological and developmental processes. A further example concerns insect viruses, which produce chitinases to disintegrate the peritrophic matrix of their organism, during the first stage of infection thereby increasing virus infectiousness.
Degradation of chitin has a number of important applications. For example, chitinases have been shown to have fungicidal activity, and to ply a role in infectivity of insect viruses. There are also several examples of transgenic plants which by expressing a foreign chitinase gene, have acquired increased resistance towards certain fungal pathogens.
Degradation products of chitin which include the monomeric sugar, N-acetylglucosamine, or oligosaccharides of N-acetylglucosamine can also be used for various different applications.
It can be seen that chitin degradation is important in a number of different fields. Although chitinase enzymes have been known for some time and have been studied in various systems, methods of improving the efficiency or the rate of degradation of chitin would be desirable. The inventors' finding that the efficiency of chitin degradation can be influenced by a chitin binding protein provides a means by which the efficiency of chitin degradation can be modulated artificially, by exposing chitin to such proteins.
In work leading up to the present invention, it has surprisingly been shown that efficient chitin degradation by chitinases in the Gram negative soil bacterium Serratia depends not only on the three Serratia chitinases, by also on the action of a small non-catalytic protein, CBP21, which binds to the insoluble crystalline chitin substrate on which its effect was tested. Although not wishing to be bound by theory, the binding of this non-catalytic protein is believed to lead to structural changes in the chitin substrate, which result in increased substrate accessibility for the chitinases, and hence to improved chitin degradation by chitinases.
Although CBP21 was identified a number of years previously (by Suzuki et al. Biosci Biotechnol Biochem. 1998 January; 62 (1): 128-35), no biological role had previously been identified for this protein. This property of CBP21 was further investigated by generating and testing CBP21 variants with single mutations on the largely polar binding surface. Various mutants lost their ability to promote chitin degradation, while retaining considerable affinity for the polymer. Thus, binding alone is not sufficient for CBP21 functionality, and this seems to depend on specific, mostly polar interactions between the protein and crystalline chitin. This is the first time that a secreted binding protein has been shown to assist in the enzymatic degradation of an insoluble carbohydrate via non-hydrolytic disruption of the substrate (Vaaje-Kolstad et al (2005a) JBC 280 (31) 28492-7 which is incorporated herein by reference).
Various homologues of CBP21 have been shown to exist in other chitin-degrading microorganisms, suggesting a general mechanism by which chitin-binding proteins function to enhance chitinolytic activity in nature. Homologues also occur in chitinase-containing insect viruses, whose infectiousness is known to depend on chitinase efficiency. No biological role had been previously identified for those homologues.
The findings made by the present inventors thus provide a means by which chitin degradation may be promoted in an in vivo, or industrial context.
The invention thus provides a method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic chitin binding protein (CBP).
Alternatively stated, the present invention provides a method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase, e.g. chitinase and a method of degrading chitin comprising exposing chitin to a non-hydrolytic CBP and chitin hydrolase, e.g. chitinase.
Chitin is defined herein as any polymer containing β(1-4) linked N-acetylglucosamine residues that are linked in a linear fashion. Crystalline chitin in the α form (where the chains run anti-parallel), β form (where the chains run parallel) or γ form (where there is a mixture of parallel and antiparallel chains), amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated & chitosan (a linear soluble polysaccharide composed of β-(1-4)-lined D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), which can be produced commercially by deacetylation of chitin) are all included within the definition of this term.
Other forms of chitin that are found in nature include copolymers with proteins and these copolymers, which include protein chitin matrices that are found in insect and crustacean cells, and any other naturally occurring or synthetic copolymers comprising chitin molecules as defined herein, are also included within the definition of “chitin”.
The term “chitin” thus includes purified crystalline α, β and γ preparation, or chitin obtained or prepared from natural sources, or chitin that is present in natural sources. Examples of such natural sources include squid pen, shrimp shells, crab shells, insect cuticles and fungal cell walls. Examples of commercially available chitins are those available from sources such as France Chitin, Hov-Bio, Sigma, Sekagaku Corp. amongst others.
Degradation of chitin is caused by the enzymatic hydrolysis of the β(1-4) bonds that like the N-acetylglucosamine residues present in chitin. Depending on the number of bonds that are hydrolysed, this results in the generation of smaller fragments of chitin, N-acetylglucosamine multimers and N-acetylglucosamine monomers. Chitinases are in general responsible for this enzymatic hydrolysis.
Irrespective of any influence that exposure to an non-hydrolytic CBP has on the bonds between chitin subunits and any other members of the copolymer that might be present, degradation of chitin as referred to herein is confined to hydrolysis of the glycoside bonds that connect the β(1-4) N-acetylglucosamine bond units in a chitin substrate.
This can be carried out by a chitinase enzyme, a chitosanase (Glycoside hydrolase families 46, 75 and 80), or a lysozyme (Glycoside hydrolase families 23 and 24). The rate or degree of hydrolysis of the glycosidic bonds that connect the β(1-4) N-acetylglucosamine bond units in a chitin substrate by any of these enzymes may be enhanced in the methods of the invention. These enzymes are referred to collectively herein as chitin hydrolases.
By “enhancing the degradation of chitin”, it is meant promoting or increasing the degree or rate of hydrolysis of the glycosidic bonds that connect the β(1-4) N-acetylglucosamine bond units of a chitin substrate, e.g. by chitinase, relative to the normal or usual degree or rate of hydrolysis of the glycosidic bonds that connect the β(1-4) N-acetylglucosamine bond units of a chitin substrate which is seen when said chitin substrate has not been exposed to a non-hydrolytic CBP. This can readily be determined by measuring the hydrolysis product formation e.g. at certain defined time points or by measuring the amount of undegraded chitin substrate which remains e.g. at certain defined time points. This can be carried out using methods that are well known in the art, based on e.g. determination of liberated reducing sugars (Horn, et al Carbohydrate Polymers, 56(1), 35-39. 2004 and references therein) or determination of liberated chitin fragments, e.g. by quantitative analysis of chromatograms obtained upon High Performance Liquid Chromatography (Hoell, I. et al (2005) Biochim Biophys Acta 1748(2), 180-190 Vaaje-Kolstad, G et al (2005a supra))
If the rate of hydrolysis, i.e. the number of bonds hydrolysed in a certain time period is greater when the chitin substrate has been exposed to non-hydrolytic CBP than when it has not, then the rate of degradation is considered to be enhanced.
Similarly, if the number of bonds that are hydrolysed in a certain time period is greater when the chitin substrate has been exposed to non-hydrolytic CBP than when it has not, then the degree of degradation is considered to be enhanced.
The rate or degree of degradation is also considered to be enhanced when the time taken for complete degradation of the chitin substrate is reduced when the chitin substrate has been exposed to non-hydrolytic CBP than when it has not. For example, preferably the use of non-hydrolytic CBP in accordance with the present invention will reduce the time taken for complete degradation by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold. In addition, the use of a non-hydrolytic CBP in accordance with the present invention can enable the complete degradation of a chitin substrate under conditions where previously complete degradation was not possible. For example, in embodiments of the invention where non-hydrolytic CBP is used in conjunction with a chitin hydrolase such as a chitinase, a chitosanase or lysozyme enzyme, then in cases where the chitin hydrolase (e.g. chitinase, chitosanase or lysozyme) enzyme alone cannot give rise to complete degradation of the substrate, the described use of a non-hydrolytic CBP can result in such complete degradation. This effect is also considered to be an enhancement of degradation.
The degree of hydrolysis is also considered to be enhanced if the overall amount of solubilisation of the chitin substrate based on exposure to a certain amount of enzyme or an enzyme cocktail is increased when said substrate has been exposed to non-hydrolytic CBP, compared to the amount that is achieved when the substrate has not been exposed to non-hydrolytic CBP.
The rate of hydrolysis is furthermore considered to be enhanced if the maximum degree of solubilization of a certain chitin-containing substrate based on exposure to a certain amount of enzyme or an enzyme cocktail is attained faster when the substrate has been exposed to non-hydrolytic CBP than when it has not been exposed to non-hydrolytic CBP.
Preferably, any such enhancement of degradation is a statistically significant one, more preferably with a probability value of <0.05. Appropriate methods of determining statistical significance are well known and described in the art and any of these may be used.
As demonstrated in the Examples, the kinetics of chitin degradation by chitinase hydrolysis of β(1-4) N-acetylglucosamine bonds when assayed in in vitro experiments are not straightforward linear kinetics. There are several kinetic phases, which in the literature are ascribed to hydrolysis of subfractions of the substrate with varying degrees of crystallinity and, thus, varying degrees of accessibility for chitinases. Sometimes, individual phases can be observed in degradation progress curves. The fast first phase is often linear and can be used to calculate initial hydrolytic rates. This phase is believed to reflect hydrolysis of the easily accessible and amorphous (i.e. the chitin chains are not orderly arranged as in a crystal, but rather disordered and thus in more contact with the solvent) regions in the substrate. During subsequent slower phases it is believed that the more recalcitrant crystalline regions of the substrate are degraded. Similar kinetics are likely to apply in respect of other chitin hydrolases.
The non-hydrolytic CBP as used herein in the methods of the invention result in a minor enhancement in the degree or rate of β(1-4) N-acetylglucosamine bond hydrolysis by the chitin hydrolase in the first, fast phase of the degradation process. However, a large effect is observed on the second slower phase. It is therefore important that if such assays with chitin hydrolases such as chitinase are performed (e.g. to determine experimentally whether a non-hydrolytic CBP has in fact functioned under the particular experimental set up to enhance the degradation of the chitin substrate, according to the definitions provided herein), a suitably long incubation time is used. As such, to detect the effect of a non-hydrolytic CBP on the rate or degree of chitin hydrolase, e.g. chitinase, degradation it is necessary to assay the rate or degree of chitin degradation after sufficiently long incubation times such that the reaction enters the subsequent, slower phase. In Example 2 (see FIG. 2) this subsequent phase is observed after the chitin (in this case β chitin), non-hydrolytic CBP (in this case CBP21) and chitinase (in this chase ChiA, B, C and G) has been incubated together for approximately 16-24 or 50 hours.
The precise kinetics will depend on many factors, such as the type of chitin, the degree of amorphousness of the chitin, the amount of enzyme present, the temperature of the pH. The degree of amorphousness will vary with the substrate source and isolation/purification process, but can be assessed by measuring the degree of crystallinity of the substrate (which is a method known in the art). Furthermore, since α-chitin is a more recalcitrant chitin form compared to the β-variant, it is anticipated that longer incubation times will be necessary in order to observe chitin degradation and hence any effect of the non-hydrolytic CBP on the rate or degree of chitin degradation.
Taking these considerations into account therefore by choosing an appropriate incubation time, the skilled person would readily be able to determine whether the degree or rate of chitin degradation is enhanced in accordance with the present invention.
The above considerations apply irrespective of the order in which the chitin is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase, i.e. if non-hydrolytic CBP and chitin hydrolase, e.g. chitinase are not administered to the chitin substrate simultaneously, or the chitin substrate is not exposed to non-hydrolytic CBP and chitin hydrolase, e.g. chitinase, together or the chitin substrate is exposed first to non-hydrolytic CBP and then to chitin hydrolase, e.g. chitinase (for example wherein the exposure to chitin hydrolase, e.g. chitinase occurs such that the chitin hydrolase, e.g. chitinase is added to the chitin and non-hydrolytic CBP or the non-hydrolytic CBP is first removed and the chitin substrate is then exposed to the chitin hydrolase, e.g. chitinase alone), the efficacy of the method can be determined in the same way.
Comparison of the rate or degree of hydrolysis of the glycosidic bonds connecting β(1-4) N-acetylglucosamine units by an appropriate chitin hydrolase, e.g. chitinase of a chitin substrate that has been exposed to non-hydrolytic CBP with the rate or degree of hydrolysis of the glycosidic bonds connecting β(1-4) N-acetylglucosamine units by an appropriate chitin hydrolase, e.g. chitinase of a chitin substrate that has not been exposed to non-hydrolytic CBP will similarly show whether or not the degradation of the chitin substrate is considered to have been enhanced in accordance with the present invention.
The methods of the invention can alternatively be viewed as a method of weakening, e.g. disrupting or interfering with or modulating the structure of a chitin substrate, comprising exposing chitin to a non-hydrolytic CBP. As noted above, and as described in the Examples, it is believed that the function of e.g. CBP21 (and other non-hydrolytic CBPs) is to interact with and affect the overall structure of chitin such that it becomes more sensitive to degradation/hydrolysis by chitinases, which may be due to improved accessibility of the chitinase substrate of chitin. The structure of chitin is preferably weakened without any hydrolysis of the β1-4 N-acetylglucosamine bonds present in said chitin.
By weakened, it is meant simply that the chitin substrate is rendered more sensitive to chitin hydrolase, e.g. chitinases, i.e. it is more easily digested or hydrolysed to chitin hydrolases, e.g. chitinases. Alternatively stated, the structure of the chitin substrate is disrupted so that a structural change can be observed by electron microscopy, or such that the mechanical strength of the chitin-containing structure is reduced, or such that a reduction in crystallinity can be observed using atomic force microscopy or electron scattering analysis.
By “non-hydrolytic CBP” (CBP=chitin binding protein) it is meant a protein that binds to chitin but that itself has no hydrolytic enzyme activity, e.g. no significant or detectable chitin hydrolase, e.g. chitinase activity (it cannot hydrolyse the β-1,4-N-acetylglucosamine linkages that are present in the chitin substrate), or not significant or detectable manganese activity (it cannot hydrolyse the β-1-4-N-mannose linkages that are present in mannan). As such, the use of whole chitin hydrolase, e.g. chitinase enzymes such as ChiA, ChiB and Chi C which have chitinase activity and contain one or more chitin binding modules is explicitly excluded. Assays to detect chitinase activity (or mannanase activity) are well known in the art. The absence of chitinase activity or mannanase activity in a CBP can therefore readily be determined.
Proteins that have the ability to bind to carbohydrates are widespread in nature. In general, such proteins contain one or more carbohydrate binding modules as part of their overall structure. These proteins may for example to be structural or singalling molecules or they can be enzymes, and the overall function of the protein may be determined by domains that are present in addition to the carbohydrate binding module. A carbohydrate-binding module (CBM) is defined as contiguous amino acid sequence within a carbohydrate binding protein with a discreet fold having carbohydrate-binding activity. Indeed, chitinases are known which contain one or more chitin binding modules in addition to catalytic regions. ChiA of Serratia Marcescens contains a fibronectin type III-type CBM, ChiB of Serratia Marcescens contains a family 5 CBM and ChiC of Serratia Marcescens contains a family 12 and a fibronectin type III-like CBM. See Y. Bourne, B. Henrissat, Curr. Opin. Struct. Biol. 11, 593 (2001) for domain nomenclature.
Proteins that a bind to carbohydrates may be classified based on function and also based on structural and/or sequence characteristics. The proteins that are of particular use in the present invention are chitin binding proteins that contain chitin binding modules that are members of family 33, according to the CAZY classification system (available on the World Wide Web at cazy.org/CAZY/fam/acc CBM.html), which is based on sequence similarities (Davies, G. J., and Henrissat, B. (2002) Biochem Soc T30, 291-297 and Y. Bourne, B. Henrissat, (2001) supra). The non-hydrolytic CBP as defined herein generally contains a carbohydrate binding module that is a member of family 33, and is thus referred to as a family 33 CBP.
All members of family 33 bind to carbohydrate structures (primarily chitin) and contain a family 33 carbohydrate binding module. In several cases, the chitin binding module makes up the whole protein, i.e. the chitin binding protein consists of or consists essentially of a single family 33 chitin binding module, that is in nature synthesised and secreted as such. However some family 33 CBMs may be fused to one or more additional non-catalytic carbohydrate binding modules (e.g. CBM family 2, CBM family 3 and CBM family 5 modules). These proteins are bi- or multi-modular non-catalytic chitin binding proteins. There is also one known example of a family 33 carbohydrate binding module that is present as an individual module within a much larger catalytic protein. This is the β1,4-mannanase protein of Caldibacillus cellulovorans (Sunna A et al (2000) Applied and Environmental Microbiology 66(2): 664-670).
The family 33 CBM is approximately 150-250 amino acids, e.g. 160-240, 170-230, 180-220, 190-210 amino acids in size and has a molecular weight of a approximately 20 kDa, preferably 19-21 kDa, 18-21 kDa, 19-22 kDa or 18-20 kDa in size. The size of a protein can readily be determined by standard methods that are known in the art.
The non-hydrolytic CBP is thus preferably a family 33 CBP. In general, the non-hydrolytic CBP contains a single family 33 chitin binding module. Preferably, the non-hydrolytic CBP consists of a single family 33 chitin binding module, or consists essentially of a family 33 chitin binding module.
If said non-hydrolytic CBP “consists essentially of” a family 33 chitin binding module, it is meant that additional amino acids may be present in the protein, in addition to those that make up the family 33 CBM. Preferably there are 1-3, 1-5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 and 90-100 or more additional amino acids present. These additional amino acids are in general present C terminal to the family 33 CBM.
Alternatively, the non-hydrolytic CBP can comprise a family 33 CBM. Additional modules or domains may thus be present in the protein. These additional modules or domains are non-catalytic or non-hydrolytic modules or domains and as such the CBP as a whole is non hydrolytic. The additional modules or domains can be CBMs. Examples of such modules are CBM family 2, CBM family 3 and CBM family 5 modules.
If additional domains or modules are present, they are in general found C-terminal to the family 33 CBM. The presence of additional domains or modules will of course increase the overall size of the protein.
The non-hydrolytic CBP can be or can comprise or correspond to a naturally occurring family 33 chitin binding module such as CBP21 (or to a homologue thereof from another species). It can alternatively be or can comprise or correspond to a variant of a naturally occurring non-hydrolytic family 33 CBP.
Naturally occurring non-hydrolytic CBPs that can be used in the invention include microbial (e.g. bacterial), eukaryotic (e.g. Dictyostelium) or viral non-hydrolytic CBPs. Bacterial non-hydrolytic CBPs are however preferred.
Examples of suitable non-hydrolytic CBPs are set out in Table 1:
BACTERIAPROTEINORGANISMGENBANK/GENPEPTCbp1Alferomonas sp. O-7AB063629BAB79619.1chitin binding protein ChbABacillus amyloliquefaciens ALKO 2718AF181997AAG09957.1BA_3348Bacillus anthracis str. A2012NC_003995NP_656708.1BA2827Bacillus anthracis str. AmesAE017033AAP26659.1NC_003997NP_845173.1BA2793Bacillus anthracis str. AmesAE017032AAP26628.1NC_003997NP_845142.1GBAA2827Bacillus anthracis str. Ames 0581AE017334AAT31944.1GBAA2793Bacillus anthracis str. Ames 0581AE017334AAT31910.1BAS2636Bacillus anthracis str. SterneAE017225AAT54946.1BAS2604Bacillus anthracis str. SterneAE017225AAT54914.1BCE2855Bacillus cereus ATCC 10987AE017273AAS41767.1NC_003909NP_979159.1BCE2824Bacillus cereus ATCC 10987AE017273AAS41736.1NC_003909NP_979128.1BC2827Bacillus cereus ATCC 14579AE017007AAP09778.1NC_004722NP_832577.1BC2798Bacillus cereus ATCC 14579AE017007AAP09751.1NC_004722NP_832550.1pE33L466_0276 (ChbA)Bacillus cereus E33LCP000040AAY60428.1BTZK2523 (ChB)Bacillus cereus ZKCP000001AAU17736.1BTZK2552 (ChB)Bacillus cereus ZKCP000001AAU17707.1ABC1161Bacillus clausii KSM-K16AP006627BAD63699.1BH1303Bacillus halodurans C-125AP001511BAB05022.1NC_002570NP_242169.1BLi00521 or BL00145Bacillus licheniformis DSM 13 ATCC 14580CP000002AAU22121.1AE017333AAU39477.1BT9727_2586 (ChB)Bacillus thuringiensis serovar konkukian str. 97-27AE017355AAT61310.1BT9727_2556 (ChB)Bacillus thuringiensis serovar konkukian str. 97-27AE017355AAT61323.1BMAA1785Burkholderia mallei ATCC 23344CP000011AAU45854.1BMA2896Burkholderia mallei ATCC 23344CP000010AAU48386.1BURPS1710b_0114Burkholderia pseudomallei 1710bCP000124ABA49030.1BURPS1710b_A2047Burkholderia pseudomallei 1710bCP000125ABA53645.1BPSL3340Burkholderia pseudomallei K96243BX571965CAH37353.1BPSS0493Burkholderia pseudomallei K96243BX571966CAH37950.1Bcep18194_C6726Burkholderia sp. 383CP000150ABB05775.1BTH_Ii1925Burkholderia thailandensis E264; ATCC 700388CP000085ABC34637.1BTH_I3219Burkholderia thailandensis E264; ATCC 700388CP000086ABC38514.1-1,4-mannanase (ManA)Caldibacillus cellulovoransAF163837AAF22274.1CV0554Chromobacterium violaceum ATCC 12472AE016911AAQ58230.1NC_005085NP_900224.1CV0553Chromobacterium violaceum ATCC 12472AE016911AAQ58229.1NC_005085NP_900223.1CV2592 (CpbD)Chromobacterium violaceum ATCC 12472AE016919AAQ60262.1NC_005085NP_902262.1CV3489Chromobaterium violaceum ATCC 12472AE016922AAQ61150.1NC_005085NP_903159.1CV3323 (CbpD1)Chromobacterium violaceum ATCC 12472AE016921AAQ60987.1NC_005085NP_902993.1EF0362Enterococcus faecalis V583AE016948AAO80225.1NC_004668NP_814154.1Sequence 4287 from patentEnterococcus faecium—AAQ43729.1U.S. Pat. No. 6583275FTL_1408Francisella tularensis subsp. holarctica LVSAM233362CAJ79847.1FTT0816cFrancisella tularensis subsp. tularensis Schu 4AJ749949CAG45449.1HCH_00807Hahella chejuensis KCTC 2396CP000155ABC27701.1HCH_03973Hahella chejuensis KCTC 2396CP000155ABC30692.1Ip_1697Lactobacillus plantarum WCFS1AL935256CAD64126.1NC_004567NP_785278.1LSA1008Lactobacillus sakei subsp. sakei 23KCR936503CAI55310.1YucGLactococcus lactis subsp. lactis IL 1403AE006425AAK06049.1NC_002662NP_268108.1Ipp0257Legionella pneumophila ParisCR628336CAH11404.1Lin2611Listeria innocuaAL596173CAC97838.1NC_003212NP_471941.1Lmo2467Listeria monocytogenes EGD-eAL591983CAD00545.1NC_003210NP_465990.1LMOf2365_2440Listeria monocytogenes str. 4b F2365AE017330AAT05205.1OB0810Oceanobacillus iheyensis HTE831AP004595BAC12766.1NC_004193NP_691731.1PBPRB0312Photobacterium profundum SS9CR378676CAG22185.1plu2352Photorhabdus luminescens subsp. laumondii TTO1BX571866CAE14645.1NC_005126NP_929598.1Sequence 6555 from patentProteus mirabilis—AAR43285.1U.S. Pat. No. 6605709chitin-binding protein ChiBPseudoalteromonas sp. S9AF007895AAC79666.1chitin-binding protein (CbpD; PA0852)Pseudomonas aeruginosa PAO1AE004520AAG04241.1NC_002516NP_249543.1chitin-binding protein (CbpD)Pseudomonas aeruginosa PAO25AF196565AAF12807.1PFL_2090Pseudomonas fluorescens Pf-5CR000076AAY91365.1Pfl_3569Pseudomonas fluorescens PfO-1CP000094ABA75307.1Psyr_2856Pseudomonas syringae pv. syringae B728aCP000075AAY37892.1PSPTO2978Pseudomonas syringae pv. tomato str. DC3000AE016866AAO56470.1NC_004578NP_792775.1RF_0708Rickettsia felis URRWXCal2CP000053AAY61559.1chitin-binding protein (CbpA)Saccharophagus degradans 2-40BK001045DAA01337.1ORFSallnivibrio costicola 5SM-1AY207003AAP42509.1chitin-binding protein (Cbp21)Serratia marcescens 2170AB015998BAA31569.1chitin-binding protein (Cbp21)Serratia marcescens BJL200AY665558AAU88202.1ORF2Serratia marcescens KCTC2172L38484AAC37123.1SO1072Shewanella oneidensis MR-1AE015551AAN54144.1NC_004347NP_716699.1SG1515 (possible fragment)Sodalis glossinidius str. ‘morsitans’AP008232BAE74790.1SAV6560Streptomyces avermitilis MA-4680AP005047BAC74271.1NC_003155NP_827736.1SAV2168Streptomyces avermitilis MA-4680AP005029BAC69879.1NC_003155NP_823344.1SAV5223 (Chb)Streptomyces avermitilis MA-4680AP005042BAC72935.1NC_003155NP_826400.1SAV2254 (CelS2)Streptomyces avermitilis MA-4680AP005030BAC69965.1NC_003155NP_823430.1SCO7225 or SC2H12.24Streptomyces coelicolor A3(2)AL359215CAB94648.1NC_003888NP_631281.1SCO6345 or SC3A7.13Streptomyces coelicolor A3(2)AL031155CAA20076.1NC_003888NP_630437.1SCO2833 (Chb)Streptomyces coelicolor A3(2)AL136058CAB65563.1NC_003888NP_627062.1SCO0643 or SCF91.03cStreptomyces coelicolor A3(2)AL132973CAB61160.1NC_003888NP_624952.1SCO0481 or SCF80.02Streptomyces coelicolor A3(2)AB017013BAA75647.1AL121719CAB57190.1NC_003888NP_624799.1SCO1734 or SCI11.23Streptomyces coelicolor A3(2)AL096849CAB50949.1NC_003888NP_626007.1CelS2 (SCO1188 or SCG11A.19)Streptomyces coelicolor A3(2)AL133210CAB61600.1NC_003888NP_625478.1chitin binding proteinStreptomyces griseusAB023785BAA86267.1cellulose binding protein (ORF2)Streptomyces halstediiU51222AAC45430.1chitin-binding proteinStreptomyces olivaceoviridis ATCC 11238X78535CAA55284.1chitin binding protein (Chb2)Streptomyces retculiY14315CAA74695.1chitin-binding protein (Cbp1)Streptomyces thermoviolaceus OPC-520AB110078BAD01591.1chitin-binding protein celS2Streptomyces viridosporusAF126376AAD27623.1Tfu_1665 (E8)Thermobifida fusca YXCP000088AAZ55700.1Tfu_1268 (E7)Thermobifida fusca YXCP000088AAZ55306.1VCA0140Vibrio cholerae N16961AE004355AAF96053.1NC_002506NP_232540.1VCA0811Vibrio cholerae N16961AE004409AAF96709.1NC_002506NP_233197.1VFA0143Vibrio fischeri ES114CP000021AAW87213.1VFA0013Vibrio fischeri ES114CP000021AAW87083.1VPA0092Vibrio parahaemolyticus RIMD 2210633AP005084BAC61435.1NC_004605NP_799602.1VPA1598Vibrio parahaemolyticus RIMD 2210633AP005089BAC62941.1NC_004605NP_801108.1VV21258Vibrio vulnificus CMCP6AE016812AAO08152.1NC_004460NP_763162.1VV20044Vibrio vulnificus CMCP6AE016808AAO07021.1NC_004460NP_762031.1VVA0086Vibrio vulnificus YJ016AP005344BAC96112.1NC_005140NP_936142.1VVA0551Vibiro vulnificus YJ016AP005346BAC96577.1NC_005140NP_936607.1ChiYYersinia enterocolitica (type 0:8) WA-314AJ344214CAC83040.2YP0706Yersinia pestis biovar Medievalis str. 91001AE017129AAS60972.1NC_005810NP_992095.1YPO3227Yersinia pestis CO92AJ414156CAC92462.1NC_003143NP_406699.1Y0962Yersinia pestis KIMAE013699AAM84543.1NC_004088NP_668292.1YPTB3366Yersinia pseudotuberculosis IP 32953BX936398CAH22604.1YPTB0899Yersinia pseudotuberculosis IP 32953BX936398CAH20139.1
EUKARYOTAPROTEINORGANISMGENBANK/GENPEPTORF-26Agrotis segetum nucleopolyhedrovirusDQ123841AAZ38192.1spheroidin-like protein (Gp 37)Autographa californica nucleopolyhedrovirusL22858AAA66694.1D00583BAA00461.1NC_001623NP_054094.1fusolinBombyx mori nuclear polyhedrosis virusU55071AAB47606.1L33180AAC63737.1NC_001962NP_047468.1spheroidinChoristoneura biennis entomopoxvirusM34140AAA42887.1
VIRUSESPROTEINORGANISMGENBANK/GENPEPTORF-26Agrotis segetum nucleopolyhedrovirusDQ123841AAZ38192.1Spheroidin-like protein (Gp 37)Autographa californica nucleopolyhedrovirusD00583BAA00461.1L22858AAA66694.1NC_001623NP_054094.1fusolinBombyx mori nuclear polyhedrosis virusU55071AAB47606.1NC_001962NP_047468.1L33180AAC63737.1spheroidinChoristoneur biennis entomopoxvirusM341140AAA42887.1ORF60Choristoneura furniferana defective nucleopolyhedrovirusAY327402AAQ91667.1NC_005137NP_932669.1spindle-like proteinChoristoneura furniferana nuclear polyhedrosis virusU26734AAC55636.1NC_004778NP_848371.1GP37 (ORF-67 GP37)Chrysodeixis chalcites nucleopolyhedrovirusAY864330AAY83998.1ORF57Epiphyas postvittana nucleopolyhedrovirusAY043265AAK85621.1NC_003083NP_203226.1GP37Helicoverpa armigera single nucleocapsid polyhedrovirusAF266696AAK57880.1AF303045AAK96305.1NC_003094NP_203613.1ORF59Helicoverpa zea nucleopolyhedrovirusAF334030AAL56204.1NC_003349NP_542682.1gp37Heliocoverpa armigera nucleopolyhedrovirus G4AF271059AAG53801.1NC_002654NP_075127.1fusolinHeliothis armigera entomopoxvirusL08077AAA92858.1HynVgp086 (slp)Hyphantria cunea nucleopolyhedrovirusAP009046BAE72375.1Gp37 proteinLeucania separata nuclear polyhedrosis virusAB009614BAA24259.1fusolin-like proteinLymantria dispar nucleopolyhedrovirusU38895AAB07702.1AF081810AAC70254.1NC_001973NP_047705.1gp37 proteinMamestra brassicae nucleopolyhedrovirusAF108960AAD45231.1ORF 37 (Gp37)Mamestra configurata nucleopolyhedrovirus AU59461AAM09145.1AF539999AAQ11056.1Gp37Mamestra configurata nucleopolyhedrovirus BAY126275AAM95019.1NC_004117NP_689207.1spheroidin-like protein (Gp 37)Orgyia pseudotsugata nuclear polyhedrosis virusU75930AAC59068.1D13306BAA02566.1NC_001875NP_046225.1enhancing factorPseudaletia separata entomopoxvirusD50590BAA09138.1ORF25Spodoptera exigua nucleopolyhedrovirusAF169823AAF33555.1NC_002169NP_037785.1gp37 (fragment)Spodoptera frugiperda MNPVAY250076AAP79107.1ubiquitin GP37 fusion proteinSpodoptera litura nucleopolyhedrovirus G2AF325155AAL01718.1NC_003102NP_258300.1gp37Trichoplusia ni single nucleopolyhedrovirusDQ017380AAZ67435.1fusolinunidentified entomopoxvirusX77616CAA54706.1ORF107Xestia c-nigrum granulovirusAF162221AAF05221.1NC_002331NP_059255.1
Bacterial non-hydrolytic CBPs can be from any appropriate source but are preferably from a species selected from the group consisting of Bacillus, Chromobacterium, Enterococcus, Francisella, Hahella, Lactobacillus, Lactococcus, Legionella, Listeria, Oceanobacillus, Photobacterium, Photothabdus, Proteus, Pseudoalteromonas, Pseudomonos, Rickettsia, Saccharophagus, Salinvibrio, Serratia, Shewanella, Sodalis, Streptomyces, Thermobifida, Vibrio and Yersinia. 
Examples of specific non-hydrolytic CBPs are CBP 21 of Serratia Marescens (SEQ ID NO: 1, MNKTSRTLLSLGLLSAAMFGVSQQANAHGYVESPASRAYQCKLQLNTQCGSVQYEPQSVEGLKGFPQAGPADGHIASADKSTFFELDQQTPTRWNKLNLKTGPNSFTWKLTARHSTTSWRYFITKPNWDASQPLTRASFDLTPFCQFNDGGAIPAAQVTHQCNIPADRSGSHVILAVWDIADTANAFYQAIDVNLSK, where amino acid residues 1 to 27 correspond to the leader peptide that is necessary for secretion of the protein in a natural system and amino acids 28-197 correspond to the mature protein), ChbA of B. amyloliquefaciens (Chu et al, Microbiology. 2001 July; 147 (Pt 7): 1793-803) CHB1, 2 & 3 of Streptomyces (Svergun et al., Biochemistry. 2000 Sep. 5; 39 (35): 10677-83, Zeltins et al., Eur J Biochem. 1997 Jun. 1; 246 (2): 557-64, Zeltins et al., Anal Biochem. 1995 Nov. 1; 231 (2): 287-94, Schnellmann et al., Mol Microbiol. 1994 September; 13 (5): 807-19, Kolbe et al., Microbioloy. 1998 May; 144 (Pt 5): 1291-7, Saito et al., Appl Environ Microbiol. 2001 March; 67 (3): 1268-73), CBP1 of Alteramonas (Tsujibo et al, Appl. Environm. Microbiol. 68: 263-270 (2002)). All of these references are incorporated herein by reference.
The non-hydrolytic CBP can thus be or correspond to or comprise a naturally occurring CBP (such as CBP21, ChbA, CHB1, 2 & 3 and CBP1) in that it is a CBP (e.g. a family 33 CBP) that is found in nature.
Alternatively, the non-hydrolytic CBP can be derived from a naturally occurring protein which contains an appropriate CBM, such as the β1,4 mannanase referred to above, or any other family 33 CBP that contains modules or domains that are present in addition to the family 33 CBM. In the latter case, the appropriate CBM for use as a non-hydrolytic CBP in accordance with the present invention can be obtained by any appropriate method. For example, the sequence that encodes the CBM can be determined and expressed in isolation from the rest of the protein using standard molecular biology techniques that are known in the art. Alternatively, for example, the CBM can be removed from the native protein by for example proteolytic cleavage.
Variants, derivatives or fragments of naturally occurring CBPs or CBMs can also be used, providing that these variants, derivatives or fragments retain the functional property of the CBP in that they bind to chitin and enhance the degradation of chitin or weaken chitin as described herein, for example by causing disruption to the chitin substrate, i.e. they should be considered to be functionally equivalent variants. As noted elsewhere herein, the property can be tested for in a straightforward manner.
The non-hydrolytic CBP as defined herein has the property of being able to bind to at least one type of chitin. It must also have the property, which has been demonstrated for CBP21, of enhancing the degradation of chitin or weakening chitin. Whether or not a protein has the ability to bind to chitin and to enhance the degradation of chitin or weaken chitin by for example causing disruption to the chitin substrate can readily be determined by straightforward assays that have been described in the art e.g. by using the assays that are set out in Examples 2-3 and Vaaje-Kolstad et al ((2005a) supra). For example the rate of chitin degradation by chitin hydrolase, e.g. Chitinase in the presence and absence of the putative non-hydrolytic CBP or following exposure to the putative non-hydrolytic CBP can be compared.
Variants include or comprise naturally occurring variants of the non-hydrolytic CBP as defined herein such as comparable proteins or homologues found in other species or more particularly variants found within other microorganisms, which have the functional properties of a non-hydrolytic CBP as defined elsewhere herein.
Variants of the naturally occurring non-hydrolytic CBPs as defined herein can also be generated synthetically e.g. by using standard molecular biology techniques that are known in the art, for example standard mutagenesis techniques such as site directed or random mutagenesis. Such variants further include or comprise proteins having at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence similarity or identity with a naturally occurring CBP or CBM at the amino acid level.
When variants are generated, it should be noted that appropriate residues to modify depend on the properties that are being sought in such a variant. In the case that a variant having the same binding or enhancing or weakening properties of the non-hydrolytic CBP is being sought, the residues are in general those residues that are not involved in the interaction of the non-hydrolytic CBP with the chitin substrate. Preferably the residues that are modified are residues that are not involved with the property of enhancing or weakening degradation exhibited by the non-hydrolytic CBP (although of course it is possible that this property could be further improved by mutating these residues).
On the other hand, it will be appreciated by a person skilled in the art that residues that are crucial for the functionality of CBP21 (see Example 4), or analogous or corresponding residues in CBP21-like proteins may be mutated with the aim of further improving the function of the non-hydrolytic CBP. This could be done by standard protein engineering techniques or by techniques based on random mutagenesis followed by screening, all techniques that are well known in the art. Attempts to improve the function of a non-hydrolytic CBP may include improving the binding and disrupting ability of this non-hydrolytic CBP towards other types of insoluble carbohydrate polymers than the polymer(s) on which the CBP acts naturally. Such alternative polymers include carbohydrate containing copolymers, e.g. protein-carbohydrate co-polymers.
For Example, the case of CBP21, several residues have been shown to be important in the binding of CBP21 to chitin and more specifically to the ability of CBP21 to enhance the degradation of chitin. Several mutations have been shown not to affect binding, but to affect the ability of CBP21 to enhance the degradation of chitin. These residues are preferably not modified relative to the wild type CBP21 sequence as set out in SEQ ID NO: 1, if the aim is to modify e.g. the stability of the non-hydrolytic CBP (for example under process conditions), but these residues may be targeted if one's aim is to improve or change the non-hydrolytic CBP functional properties.
A person skilled in the art will recognize the potential of using the CBP framework to create variants that are optimised for other insoluble polymeric polysaccharide substrates (e.g. other forms of chitin or cellulose), or insoluble carbohydrate-containing co-polymers.
Preferred variants of CBP21 retain one or more and preferably all of: a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an asparagine at position 185 (sequence numbering according to SEQ ID NO: 1).
Further preferred variants of CBP21 retain one or more and preferably all of a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114 and an aspartic acid residue at position 182 (sequence number according to SEQ ID NO: 1).
In connection with amino acid sequences, “sequence similarity”, preferably “sequence identity”, refers to sequences which have the stated value when assessed using e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids). Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.
Preferred “variants” include those to which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analogue thereof. Amino acids used in the sequence may also be derivatized or modified, e.g. labelled, glycosylated or methylated, providing the function of the non-hydrolytic CBP is not significantly adversely affected.
Variants refers to peptides related to, or derived from the above described amino acid sequences, where the amino acid sequence has been modified by single or multiple amino acid (e.g. at 1 to 10, e.g. 1 to 5, preferably 1 or 2 residues) substitution, addition and/or deletion or chemical modification, including deglycosylation or glycosylation, but which nonetheless retain functional activity, insofar as they bind to chitin and enhance the degradation of chitin.
Within the meaning of “addition” variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide or other molecule fused to the non-hydrolytic CBP sequence. Carboxyl terminal fusions are preferred. It must of course be ensured that any such fusion to non-hydrolytic CBP does not adversely affect the functional properties required for use in the methods of the invention as set out elsewhere herein.
“Substitution” variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative substitutions. Such functionally-equivalent variants mentioned above include in particular naturally occurring biological variations (e.g. found in other microbial species) and derivatives prepared using known techniques. In particular functionally equivalent variants of the non-hydrolytic CBPs described herein extend to non-hydrolytic CBPs which are functional in (or present in), or derived from different genera or species than the specific CBP molecules mentioned herein.
Variants such as those described above can be generated in any appropriate manner using techniques which are known and described in the art, for example using standard recombinant DNA technology.
Derivative of non-hydrolytic CBPs as defined herein my also be used. By derivative it is meant a non-hydrolytic CBP or variant thereof which instead of the naturally occurring amino acid, contains a structural analogue of that amino acid. Derivatisation, modification (e.g. labelling, glycosylation, methylation) may also occur as long as the function of the non-hydrolytic CBP is not adversely affected.
Fragments of the non-hydrolytic CBPs may also be used as long as the function of the non-hydrolytic CBP required for use in the present invention is not adversely affected by the deletion of one or more amino acids from said non-hydrolytic CBP, i.e. the ability of the non-hydrolytic CBP to bind to chitin and also to enhance the degradation of chitin for example by causing disruption to the chitin substrate.
The fragment may be a truncated version of a non-hydrolytic CBP, e.g. CBP 21 e.g. C terminal deletion of 1, 2, 3, 4, 5, amino acids although in general N terminal deletions of CBP21 should be avoided.
In the most preferred embodiment, the non-hydrolytic CBP is or comprises the CBP21 protein from Serratia marcescens having the amino acid sequence set out in SEQ ID No:1. Functional variants, derivatives and fragments of this protein are also preferred.
By “exposing” the chitin to the non-hydrolytic CBP, it is meant that the non-hydrolytic CBP and chitin are brought into contact in an appropriate context and under appropriate conditions so as to allow the two components to interact or bind to each other. It has been shown for CBP21 that for this non-hydrolytic CBP, binding to chitin is necessary for its effect in enhancing chitin degradation (Vaaje-Kolstad et al 2005a, supra) and it is believed that the enhancement of chitin degradation depends on the formation of the appropriate bonds between these two components.
The chitin substrate is thus mixed with, or contacted with the non-hydrolytic CBP under suitable conditions so as to allow this interaction to take place. Suitable contact must therefore be made between the non-hydrolytic CBP and the chitin substrate so as to allow these two components to interact. Thus, the non-hydrolytic CBP may simply be brought into contact with the chitin substrate for example by adding it directly to the chitin substrate. Conveniently, the non-hydrolytic CBP may be present in a liquid medium which is applied to the chitin substrate. In general, the non-hydrolytic CBP will be present in an aqueous solution, although any appropriate conditions can be used. The following description sets out conditions that can be used, but it should be noted that any appropriate conditions can be used, and the step of “exposing” the chitin substrate to the non-hydrolytic CBP can take place in any appropriate context, e.g. in a transgenic plant as discussed in more detail below, or in the context of a medical or agricultural treatment, where the non-hydrolytic CBP is administered to a patient or a plant.
In a preferred embodiment the non-hydrolytic CBP is present in a buffer such as a phosphate buffer, e.g. a sodium phosphate buffer. Suitable concentration ranges for such a buffer are 1-100 mM. The non-hydrolytic CBP can be present in the solution at any suitable concentration, such as a concentration of 0.001-1.0 mg/ml, e.g. 0.01-0.1 mg/ml or 0.05-0.5 mg/ml.
Preferably the chitin substrate is exposed to the non-hydrolytic CBP, e.g. the chitin substrate and the non-hydrolytic CBP are incubated with each other, for a period of 12 or 24 hours or more, e.g. 36 or 48 hours or more, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. This incubation is in general carried out at or about 37° C., although appropriate temperatures for optimizing the enhancement of chitin degradation can readily be determined by the skilled person in the art. For example, the temperature can be in the range of 36-38° C. or 35-38° C.
It will be appreciated by the person skilled in the art that the necessary incubation times, pH, temperature, substrate concentrations and CBP21 concentrations are not independent of each other. Thus, a large range of conditions can be envisaged, which can easily be evaluated by a person skilled in the art.
Preferably the chitin substrate is exposed to the non-hydrolytic CBP, e.g. the chitin substrate and the non-hydrolytic CBP are incubated with each other, at an appropriate pH, for example at a pH in the range of 5.5 to 7, or 6 to 6.5, although appropriate pHs for optimizing the enhancement of chitin degradation can readily be determined by the skilled person in the art. Preferably the pH is in the range of 6.2-6.4 or 6.25-6.35. The preferred pH is about pH 6.3.
Preferably the incubation is carried out without agitation, i.e. without stirring or disturbing the mixture. This may reduce any adherence of chitin substrate to the walls of the vessel.
The exposure of the chitin substrate to the non-hydrolytic CBP can occur in vitro in any appropriate vessel, e.g. by mixing together the chitin substrate and the non-hydrolytic CBP in an appropriate medium (e.g. a solution, such as an aqueous solution) or by applying the non-hydrolytic CBP to the chitin substrate (e.g. by applying the non-hydrolytic CBP in a solution to a chitin substrate).
As indicated above, in an alternative embodiment of the invention a chitin hydrolase, e.g. chitinase, chitosanase or lysozyme may be used in combination with the non-hydrolytic CBP under appropriate conditions to enhance chitin degradation. Thus, the chitin substrate can be exposed simultaneously to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase, chitosanase or lysozyme or else the chitin substrate is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase, chitosanase or lysozyme sequentially, in either order. It is preferred that the CBP is added before the chitin hydrolase, e.g. chitinase, chitosanase or lysozyme.
Any chitin hydrolase, e.g. chitinase, chitosanase or lysozyme that is able to degrade the glycosidic bonds in the chitin substrate may be added. Such degradation may be complete or partial. For example, the activity of some chitin hydrolase, e.g. chitinases on chitin substrates is not strong enough to result in complete degradation of the substrate. This is particularly the case for chitinases such as ChiG from Streptomyces coelicolor that do not have their own CBM, or chitinases such as ChiB. In this case, the use of a non-hydrolytic CBP in accordance with the present invention can result in enhanced chitin degradation and preferentially result in complete degradation that was not previously possible. Examples of appropriate chitinases include all enzymes and putative enzymes listed in the CAZY database under glycoside hydrolase families 18 and 19.
In addition, chitosanases (glycoside hydrolase families 46, 75 and 80) and lysozymes (glycoside hydrolase families 23 and 24) may be appropriate.
Other enzymes may also be added in addition to or as an alternative to the chitin hydyrolytic enzymes discussed above, depending on the nature of the chitin substrate that is to be degraded. For example, if the chitin to be degraded is a copolymer which contains protein, proteases may also be added. Suitable examples include Alcalase, Neutrase, Papain and other broad-specificity proteolytic enzymes. In each experimental set-up the suitability of proteases will need to be checked, especially if other enzymes (e.g. chitinases), which may be destroyed by some of the available proteases, are present simultaneously. It must be noted that disruption of chitinous parts of a chitin-containing co-polymers may facilitate enzymatic degradation of the non-chitinous parts, even if no chitinase is present simultaneously. If the non-chitinous parts consist of non-protein compounds, other appropriate enzymes may be added.
In the embodiments including the use of chitin hydrolase, e.g. chitinases, the chitin substrate is mixed with, or contacted with non-hydrolytic CBP and chitin hydrolase, e.g. chitinase under suitable conditions so as to allow the appropriate interaction or interactions to take place between the chitin hydrolase, e.g. chitinase enzyme and the chitin substrate and between the non-hydrolytic CBP and the chitin substrate the enzymatic hydrolysis of the β1-4 N-acetyl glucosamine bonds to occur. As discussed above, in relation to the contact between the CBP and the chitin substrate, suitable contact must be made between the components of this system. Thus, the non-hydrolytic CBP and chitin hydrolase, e.g. chitinase may simply be brought into contact with the chitin substrate for example by adding them directly in the chitin substrate. Conveniently, the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase may be present in a liquid medium which is applied to the chitin substrate. In general, the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase will be present in an aqueous solution, although any appropriate conditions can be used.
In a preferred embodiment the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase are present in a buffer such as a phosphate buffer, e.g. a sodium phosphate buffer. Suitable concentration ranges for such a buffer are 1-100 mM. The non-hydrolytic CBP can be present in the solution at any suitable concentration, such as a concentration of 0.001-1.0 mg/ml, e.g. 0.01-0.1 mg/ml or 0.05-0.5 mg/ml.
Preferably the chitin substrate is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase e.g. the chitin substrate and the non-hydrolytic CBP are incubated with each other, for a period of 12 or 24 hours or more, e.g. 36 or 48 hours or more, or 3, 4, 5, 6, 6, 8, 9, 10, 11, 12, 13, 14 days or more. This incubation is in general carried out at or about 37° C., although appropriate temperatures for optimizing the enhancement of chitin degradation can readily be determined by the skilled person in the art. For example, the temperature can be in the range of 36-38° C. or 35-39° C.
It will be appreciated by the person skilled in the art that the necessary incubation times, pH, temperature, substrate concentrations and CBP21 concentrations are not independent of each other. Thus, a large range of conditions can be envisaged, which can easily be evaluated by a person skilled in the art.
Preferably the chitin substrate is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase, e.g. the chitin substrate, the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase are incubated with each other, at a pH in the range of 5.5 to 7, or 6 to 6.5, although appropriate pHs for optimizing the enhancement of chitin degradation can readily be determined by the skilled person in the art. Preferably the pH is in the range of 6.2-6.4 or 6.25-6.35. The preferred pH is about pH 6.3.
Preferably the incubation is carried out without agitation, i.e. without stirring or disturbing the mixture. This may reduce any adherence of chitin substrate to the walls of the vessel.
The exposure of the chitin substrate to the chitin hydrolase, e.g. chitinase and the non-hydrolytic CBP can occur in vitro in any appropriate vessel, e.g. by mixing together the chitin substrate and the chitin hydrolase, e.g. chitinase and the non-hydrolytic CBP in an appropriate medium (e.g. a solution, such as an aqueous solution) or by applying the chitin hydrolase, e.g. chitinase and the non-hydrolytic CBP to the chitin substrate (e.g. by applying the chitin hydrolase, e.g. chitinase and the non-hydrolytic CBP in a solution to a chitin substrate).
If the chitin is exposed first to the non-hydrolytic CBP and then to the chitin hydrolase, e.g. chitinase (i.e. the chitin substrate is not simultaneously exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase), the chitinase may simply be added to the chitin that has been exposed to the CBP, without the removal of the non-hydrolytic CBP, e.g. by supplementing the non-hydrolytic CBP containing solution with chitinase or mixing a chitinase containing solution with the non-hydrolytic CBP containing solution.
Alternatively, the non-hydrolytic CBP, e.g. in a non-hydrolytic CBP containing solution, may be removed from the chitin substrate and replaced with a chitin hydrolase, e.g. chitinase containing solution. In each case, the chitin is exposed first to the non-hydrolytic CBP and then to the chitinase.
Appropriate forms of non-hydrolytic CBP and variants etc., thereof, for use in the methods of the present invention are described above. Thus, the non-hydrolytic CBP that is present, e.g. in the solution, can be in many different forms, and isolated, extracted or purified from various different sources or synthesised by various different means.
For example, the non-hydrolytic CBP (for variant, etc., thereof) can be a synthetic non-hydrolytic CBP that has been chemically synthesised. Chemical syntheses may be performed by methods well known in the art involving, in the case of peptides, cyclic sets of reactions of selection deprotection of the functional groups of a terminal amino acid and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups. Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art, such as the well known Merrifield solid phase synthesis procedure. Preferably the non-hydrolytic CBPs for use in the invention are substantially purified, e.g. pyrogen-free, e.g. more than 70%, especially preferably more than 90% pure (as assessed for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing or chromatography). Purification may be performed for example by chromatography (e.g. HPLC, size-exclusion, ion-exchange, affinity, hydrophobic interaction, reverse-phase) or capillary electrophoresis.
Recombinant expression of proteins is also well known in the art and an appropriate nucleic acid sequence can be used to express a non-hydrolytic CBP (or variant, etc., thereof) for subsequent expression and optional purification using techniques that are well known in the art. For example, an appropriate nucleic acid sequence can be operably linked to a promoter for expression of the non-hydrolytic CBP (or variant, etc., thereof) in bacterial cells, e.g. E. coli. It may be convenient to express the non-hydrolytic CBP under the control of an inducible promoter, in which case the expression of the non-hydrolytic CBP will be induced in culture to cause expression.
As the non-hydrolytic CBPs are in general secreted, it is generally possible to purify them from the supernatant or periplasmic fractions of host cells using techniques that are known in the art (e.g. as described in Vaaje-Kolstad et al) to provide the source of the non-hydrolytic CBP. The purified protein can then be mixed with an appropriate liquid or solution for administration or addition to the chitin substrate that is to be exposed thereto. This approach is equally applicable to recombinant bacteria and to bacteria that naturally synthesise and secrete non-hydrolytic CBPs.
These proteins are secreted and in general contain a leader sequence that is responsible for secretion. Optionally therefore, genes encoding leader peptide-free non-hydrolytic CBPs may be fused to DNA sequences endcoding leader peptides of choice to ensure efficient secretion by certain host bacteria. Alternatively, CBP genes may be clones without leader peptide and produced intracellularly.
Alternatively, the supernatant or growth medium in which the bacteria which naturally produces the non-hydrolytic CBP or which is expressing a recombinant non-hydrolytic CBP can be used as the source of the non-hydrolytic CBP. If the bacteria naturally express the non-hydrolytic CBP, it may be necessary to first induce the secretion of the CBP by growing or culturing the bacteria in the presence of a suitable inducer of synthesis such as chitin.
Partial purification or concentration of the non-hydrolytic CBP from the supernatant or growth medium in which the bacteria which naturally produces the non-hydrolytic CBP or which is expressing a recombinant non-hydrolytic CBP is growing is also possible. Suitable examples of how this might be carried out to achieve the required concentration or purity of non-hydrolytic CBP are well known in the art. Chitin degrading organisms can be grown in media containing chitin as the only carbon source. Secreted proteins can be harvested by precipitating all the proteins present in the medium, followed by renaturation and isolation through general protein separation methods (e.g. electrophoresis). A second option is to collect the chitin from the growth medium after a period of growth, an then elute all bound proteins (which should be chitinases and chitin binding proteins) off the chitin. This can be done by first washing the chitin with a wash buffer (e.g. 50 mM sodium phosphate, pH 6.3) and then eluting the chitin bound proteins off with an elution buffer (a buffer either low or high in pH or with a high salt concentration). The protein eluted from the chitin can the be separated by standard protein separation methods.
The appropriate non-hydrolytic CBP to be used in these methods depends on the particular type of chitin or chitin containing copolymer that is to be degraded and can readily be determined by a person skilled in the art. For example, CBP21 binds only to β-chitin and would therefore by an appropriate non-hydrolytic CBP to use if the methods of the invention were to be applied to β-chitin.
ChbB from B amyloliquifaciens as described in Chu et al (supra) also binds to β-chitin and would therefore also be an appropriate non-hydrolytic CBP to use if the methods of the invention were to be applied to β-chitin.
CHB1, CHB2 and CHB3 have all been isolated from S olivaceovirides (Svergun et al, Zeltins et al, Schnellman et al Kolbe et al, Saito et al supra). The binding preferences of these three proteins have been determined and CHB1 and CHB2 bind preferably to α-chitin, whereas CHB3 binds to both α and β chitin. CBP1 from Alteromonas as described by Tsujibo et al binds to both α and β chitin, with a preference for the α form.
It is clear that a suitable or appropriate non-hydrolytic CBP to use in the methods of the invention has to be determined based on the ability of the non-hydrolytic CBP to interact with and disrupt the chitin substrate that is to be degraded, and that the current choice of the non-hydrolytic CBP for a particular chitin substrate is important. As described above, different non-hydrolytic CBPs bind to different chitin substrates, and binding is essential for the non-hydrolytic CBP to enhance the degradation of chitin.
A first step in identifying an appropriate non-hydrolytic CBP for use in the method is therefore to determine what non-hydrolytic CBPs bind to the chitin substrate of interest. This can be carried out by referring to binding studies that have been carried out in the prior art, e.g. if the nature of the chitin substrate that is to be degraded has been characterised. Alternatively, appropriate binding studies can readily be performed by the skilled man using techniques that are known in the art.
Such binding studies can also be performed if the nature of the chitin substrate that is to be degraded has not bee fully characterised. Again, this can readily be performed by the skilled man using techniques that are known in the art.
Those non-hydrolytic CBPs that bind to the chitin substrate that is to be degraded can then be tested for their ability to enhance the degradation of that substrate e.g. using assays as set out in the Examples. Modifications to the assays may be required depending on the particular chitin substrate that is to be degraded. For example, very long incubation times and/or higher enzyme concentrations are required if the substrate is α chitin, since the crystalline part of this chitin variant is very recalcitrant compared to β chitin or amorphous chitin.
It is similarly possible to use these techniques to identify whether a variant of a non-hydrolytic CBP would be suitable for use in the methods of the invention.
As discussed above, multiple chitinases exist in nature and they vary in their activity towards different chitin substrates. As such, the particular chitinase that is to be used in the methods of the invention can also be selected based on its ability to degrade the particular chitin substrate that is to be degraded. The properties of chitinases have been documented (e.g. Hollis, T et al (1997) Arch. Biochem. Biophys. 344, 335-342 and Suzuki, K., et al (1998) Biosci. Biotech. 62, 128-135] and chitinases can be tested based on routine assays that are well known to the person skilled in the art. Any type of chitinase e.g. a naturally occurring, recombinant, or synthetic chitinase may be used. Preferred chitinases are microbial chitinases and plant chitinases.
It is clear from the specific example of CBP21 that a single non-hydrolytic CBP can influence the degradation of a chitin substrate by multiple chitinases e.g. chitinases belonging to family 18 of glycoside hydrolases (e.g. ChiA, B, C) or family 19 of glycoside hydrolases (e.g. ChiG) and thus such various different chitinases or combinations of chitinases can be used with any one non-hydrolytic CBP in the methods of the invention. With any chitin substrate that is to be degraded, once a suitable non-hydrolytic CBP has been identified, one or more chitinases may be chosen to hydrolyse the glycosidic bonds connecting the β(1-4) N-acetylglucosamine sugars.
Preferred combinations are CBP21 (or variants, fragments or derivatives thereof) with one or more of ChiA, ChiB, ChiC and ChiG. This is a preferred combination for the degradation of chitin substrates that contain or consist of β chitin. For alpha chitin, a preferred combination would consist of CHB1 or CHB2 (Svergun et al, Zeltins et al, Schnellman et al Kolbe et al supra) or another alpha-chitin binding CBP21 analogue, combined with one or more of ChiA, ChiB, ChiC and ChiG. In principle, any chitinase could be tried in combination with any CBP that targets the “correct substrate”.
More than one, i.e. multiple chitin hydrolase, e.g. chitinases can be used to hydrolyse the glycosidic bonds connection the β(1-4) N-acetylglucosamine sugars. Multiple chitin hydrolase, e.g. chitinases can be added simultaneously or sequentially to the chitin substrate. Preferably 2, 3, 4, 5 or 6 or more different chitin hydrolase, e.g. chitinases can be used.
It is also possible to use more than one non-hydrolytic CBP in the methods of the invention. In other words, the chitin substrate that is to be degraded can be exposed to one or more non-hydrolytic CBP or to multiple (e.g. 2, 3, 4 or more) non-hydrolytic CBPs. The non-hydrolytic CBPs may have binding specificity to and function on the same type of chitin, or they may have different binding specificities and functions from each other. The latter may be appropriate if a chitin substrate that is to be degraded contains more than on type of chitin (for example γ chitin).
Based on the above considerations, it can be seen that a further embodiment of the invention relates to an assay method for identifying a non-hydrolytic CBP, or determining whether a protein is a non-hydrolytic CBP. In such an assay method the putative noon-hydrolytic CBP is exposed to chitin e.g. under conditions that are described above and the effect of exposure to this protein is assessed, e.g. as described above. If chitin is weakened or an improvement in the rate or degree of chitin degradation is observed after exposure to this protein, compared to in the absence of the exposure to the protein, the protein is considered to be non-hydrolytic CBP.
This invention has applications in a number of different fields and, due to the ability of the non-hydrolytic CBP to enhance chitin degradation, can be used to improve any method which is based on the action of chitinolytic enzymes (chitin hydrolase enzymes).
Thus, the methods of the invention can be used to improve or enhance chitin degradation in the conversion of a chitin-containing biomass to chitin fragments or N-acetylglucosamine.
N-acetylglucosamine has several applications (e.g. as discussed below) and thus the methods of the invention can be used to produce GlcNac for use in any of these applications. Currently, ClcNAc is most often produced by acid hydrolysis of chitin (a linear polymer of GlcNAc) extracted from crab and shrimp shells. The methods of the present invention thus provide an alternative and improved method of producing GlcNac. Similarly, chitin fragments (chito-oligosaccharides) also have a variety of applications (e.g. as discussed below), and thus the methods of the invention can be used to produce chitin fragments (chito-oligosaccharides) for use in any of these applications.
As indicated above, GlcNAc has several known applications. For example, N-acetylglucosamine and its derivative glucosamine are sold as a health product, e.g. as a food supplement. Although the actions of supplemental glucosamine have yet to be clarified, it is thought to play a role in the promotion and maintenance of the structure and function of cartilage in the joints of the body. It may also have anti-inflammatory properties. For example, the glucosamine-containing glycosaminoglycan hyaluronic acid is vital for the function of articular cartilage.
In addition, during the progression of osteoarthritis, exogenous-glucosamine may have a beneficial role. Glucosamine has also been found to have antioxidant activity and to be beneficial in animal models of experimental arthritis. Glucosamine may be used for the treatment and prevention of osteoarthritis. Glucosamine may be used for the treatment and prevention of osteoarthritis, either by itself or in combination with chondroitin sulfate.
As referred to above, fragments of chitin and their derivatives have a variety of applications, (M. G. Peter, in Biopolymers, Vol. 6: Polysaccharides II (A. Steinbüchel, Ed.), Weinheim: Wiley VCH, 2002, pp. 481-574). For example, they have been shown to act as immune stimulants, and to cause chemotactic migration of polymorphonuclear cells. They are also known to elicit defence responses in plants and function as signalling molecules in certain cellular processes in humans (e.g. stimulation of bone cell growth). See also Sven Bahrke et al, Biomacromolecules 3:696-704 (2002) and reference therein.
Fragments resulting from the degradation of chitin may be used directly or they may be used as building blocks to generate for example biologically active glycoconjugates. Biological activities of chitin fragments include roles as signalling molecules, for example in plants and mammals, e.g. humans. Other roles include immune stimulation, promotion of bone cell (chondrocyte) growth, morphogenetic activity and elicitation of defence response in plants. Certain fragments may inhibit chitinases, before or after chemical functionalization. Chitinase inhibitors are of interest because many plague organisms (malaria parasite, fungi, nematodes) need chitinases for growth.
Chitin fragments are good building blocks for synthesizing compounds that potentially interfere with enzymes involved in chitin catabolism or metabolism. Since many plague organisms contain chitin whereas humans do not, chitin catabolism and metabolism are very interesting target areas for development of drugs, fungicides, pesticides. The use as building blocks means that one may couple fragments resulting from the degradation of chitin to other compounds in order to give them a desirable activity. One example is a synthetic chitinase inhibitor described in detail by Vaaje-Kolstad et al. (Interactions of a family 18 chitinase with the designed inhibitor HM508 and its degradation product, chitobiono-delta-lactone; J Biol Chem. 2004 Jan. 30; 279 (5): 3612-9). Here, a dimer of GlcNAc has been made active as a chitinase inhibitor by coupling it to another chemical group.
As noted above, since many plague organisms contain chitin whereas humans do not, chitin catabolism and metabolism are very interesting target areas for development of drugs, fungicides, pesticides. Thus, the disruption of the cell walls and/or membranes and/or skeletons of appropriate pathogens and parasites has been a useful therapeutic strategy against pathogens (in particular fungi) and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Various anti-fungal therapeutics have been developed but fungal infections of mammals, in particular humans, have increasingly become responsible for life-threatening disorders. Furthermore, toxicity associated with known antifungal drugs can cause serious adverse side effects, and mortality rates of certain fungal infections such as systemic candidiasis remain high despite Amphotericin B treatment.
Examples of fungal species and parasites include Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
Fungi also present a serious problem to farmers as they are very common causative agents of infectious disease of crop plants. Phytopathogenic fungi cause devastating epidemics, and cause annual crop yield losses and crop failures. The effects of fungi are not specific to a few plant species, but attack all of the known species of flowering plants, and also to attack crop plants and trees.
Traditionally the strategies that are used to control plant disease include the use of disease resistant cultivars. Such cultivars can be selected or developed by plant breeders e.g. by breeding to incorporate natural resistance mechanisms into the crops.
There are however disadvantages with this approach as the genetic sources of this natural resistance are often associated with other undesirable properties and in order to recreate the desired background, extensive backcrossing and introgression is needed. This is compounded by the fact that the resistance mechanism(s) are often polygenic. In view of these problems, the steps of improving disease resistance by conventional breeding are expensive in terms of both time and money.
As an alternative to conventional breeding techniques, the use of recombinant DNA technology has provided the possibility of introducing disease resistance using transgenes. For example, an isolated resistance gene or other useful gene in plant defence mechanisms can be used to transform a plant.
This new technology has not however prevented crop disease epidemics and additional strategies should be contemplated to supplement the conventional breeding method and existing genetic engineering strategies.
As noted above, chitin is present in fungal cell walls and the walls and membranes of other pathogens and parasites, and chitinases have antibiotic action against such organism. It should therefore be possible to combat plant fungal infections and diseases in plants caused by other chitin containing pathogens, e.g. insects or nematodes by taking advantage of the newly identified properties of non-hydrolytic CBPs to improve or enhance chitin degradation or to weaken chitin.
Therefore in a further aspect, the present invention relates to a composition comprising a non-hydrolytic CBP as defined herein and a pharmaceutically or agriculturally acceptable carrier, diluent or excipient. Said compositions (and indeed other non-hydrolytic CBP containing compositions as defined herein) can be used to enhance the degradation of chitin or to weaken chitin. Said uses can be carried out in vitro, e.g. in a bioreactor or test tube, or in vivo, e.g. in a plant, animal, microorganism, etc, i.e. in any environment where chitin is present.
As the composition contains a non-hydrolytic CBP, the ability of this protein to enhance the degradation of chitin, which is key component of fungi (and other plant pathogens such as nematodes and insects), will adversely affect the growth of said fungi (and other plant pathogens such as nematodes and insects).
In addition to a non-hydrolytic CBP, the composition may further comprise other active ingredients such as chitin hydrolase, e.g. chitinase as defined herein and/or a further fungicidal or pathicidal agent. Suitable agents for inclusion in a composition that is to be used in the treatment of mammals will be known to a person skilled in the art and can be selected depending on the nature of the fungus or other pathogen which is to be treated by the composition. Suitable fungal agents include amphotericin B, nystatin, pimaricin, 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and tioconazole; allylamines-thiocarbamates such as tolnaftate, naftifme and terbinafme; griseofulvin; ciclopirox olamine; haloprogin; undecylenic acid; and benzoic acid. The fact that synergistic anti-fungal effects may be obtained by combining the applicant of chitinolytic enzymes with application of anti-fungal agents such as gliotoxin, flusilazole, miconazole, captan and benomyl is documented in the literature (Lorito M, et al Microbiology. 1994 March; 140 (Pt 3): 623-9).
Suitable additional fungicidal agents for inclusion in a composition that is to be used in the treatment of plants will be known to a person skilled in the art and can be selected depending on the nature of the fungus which is to be treated by the composition. Suitable agents include fungal cell membrane affecting compounds selected from the group consisting of sterollsynthesis inhibiting fungicides, antifungal peptide antibiotics, zeamatin and proteins that are serologically related to zeamatin, and antifungal lipid lytic enzymes. Alternative additional components of the composition include antifungal polyene macrolide antibiotics, antifungal epithiodiketopiperizine antibiotics, fungal cell wall biosynthesis inhibitors such as chitin synthetase inhibitors and β glucan synthetase inhibitors. Such compounds are described in U.S. Pat. No. 6,512,166 which is incorporated herein by reference. Such compositions can be formulated according to any of the conventional methods known in the art and widely described in the literature. Thus, the active ingredient (i.e. a non-hydrolytic CBP) may be incorporated, optionally together with other active substances (such as chitin hydrolase, e.g. chitinases and/or other fungicidal or pathicidal agents, examples of which are as described above), with one or more conventional carriers, diluents and/or excipients appropriate for the particular use for a composition, e.g., agriculturally acceptable carriers for agricultural uses and pharmaceutically acceptable carriers for medicinal uses, to produce conventional preparations which are suitable or can be made suitable for administration such as powders, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, ointments, sterile injectable solutions, sterile packaged powders, and the like. They may be formulated as liquids (solutions or suspensions) or as solids. Preferably the pharmaceutical composition comprising the non-hydrolytic CBP is prepared in a form appropriate for topical application.
Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, maltose, glucose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, polyvinylpyrrolidone, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient or plant by employing procedures well known in the art.
As described above, preferably the composition is in a form suitable for topical administration and suitable carriers may be present at any appropriate concentration, but exemplary concentrations are from 1% to 20% and preferably from 5% to 10%.
Suitable doses of the non-hydrolytic CBP and the other active ingredients (if included) will vary from plant to plant and from patient to patient and will also depend on the nature of the particular infection (as the type of chitin or co-polymer may be different). Suitable doses can be determined by the person skilled in the art or the physician in accordance with the weight, age and sex of the patient, the severity of the fungal infection, and according to the nature and age of the plant to be treated Exemplary unit doses are: 0.1 to 50 mg, of the active ingredient. Unit doses will normally be administered once or more times a day, for example 2, 3 or 4 times a day, more usually 1 to 5 times a day, such that the daily dose is normally in the range of 0.1 to 50 mg, for example 0.1 to 5 mg for a 70 kg adult, that is in the range of approximately 0.001 to 1 mg/kg/day, more usually 0.005 to 0.2 mg/kg/day.
If chitin hydrolase, e.g. chitinase is present in the composition, since free water is needed for their activity, water must be present at the time of function. This can be accomplished, for example, by making up the chitin hydrolase, e.g. chitinases as aqueous solutions or by making a formulation as a dry powder and applying the powder with the enzyme becoming active once water becomes available, e.g., from rain or saliva. Water is thus a preferred vehicle where components are soluble in it. Organic solvents can also be used and may be required in some cases if a solution is desired. Suspensions can also be employed. Most polyene macrolide antibiotics have poor water solubility and are therefore normally formulated as dispersions or suspensions for application or applied as a powder. The chitin synthetase inhibitors may also be applied as a powder.
For medicinal purposes (i.e., human and veterinary therapy) the composition comprising a non-hydrolytic CBP is preferably administered topically e.g. to the skin of a human or non-human animal. Administration can also be, at least in some instances, via parenteral injection, e.g., intraperitoneally; this administration route is particularly useful where the immune system has been compromised since immune-deficient humans and individuals will inactivate enzymatic proteins more slowly than normal individuals.
Thus, a yet further aspect of the invention provides a method for treating or preventing disease in a plant, wherein said disease is caused by a chitin containing microorganism or pathogen, for example a fungus, insect or nematode, comprising contacting the chitin containing microorganism or pathogen or contacting the plant to be protected with an effective amount of non-hydrolytic CBP as defined herein.
A preferred embodiment of the invention provides a method for treating or preventing fungal infection in a plant, comprising contacting the fungus or a plant to be protected from the fungus with an antifungal effective amount of a non-hydrolytic CBP as defined herein.
Any of the compositions that are described above can be used in such a method.
The compositions and method of the invention can be used to prevent or treat any disease in a plant which is caused by a chitin containing microorganism or pathogen.
A yet further aspect of the invention provides a method for treating or preventing disease in an animal, wherein said disease is caused by a chitin containing microorganism or pathogen, for example a fungus, insect or nematode, comprising administering to a subject in need of such a treatment with an effective amount of a non-hydrolytic CBP as defined herein.
A preferred embodiment of the invention provides a method for treating or preventing fungal infection in an animal, comprising administering to a subject in need of such a treatment an antifungal effective amount of a non-hydrolytic CBP as defined herein.
Any of the compositions that are described above can be used in such a method. The modes of administration of the non-hydrolytic CBP can be any appropriate mode as discussed above in connection with the composition.
The method is generally carried out in a mammal. Any mammal may be treated, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkeys. Preferably however the mammal is human.
The composition and method described above can be used to prevent or treat fungal infection wherein the fungal species is from genera including Fusarium, Gliociadium, Rhizoctonia, Trichoderma, Uncinula, Ustilago, Erysiphe, Botrytis, Saccharomyces, Sclerotium, Candida, Aspergillus and Alternaria. 
The composition and method described above can also be used to prevent infection or damage by insects and nematodes, e.g. nematodes belonging to the genera Globodera, Meloidogyne and Heterodera. Nematode eggs are of special interest because they contain a chitin layer in their shells. Strategies aimed at insects should normally focus on chitinous layers in the gastrointestinal tract and on the cuticle.
As used herein “treating” refers to the reduction or alleviation, preferably to normal levels, of one or more of the symptoms of disease (e.g. fungal infection) For example, such treatment might result in e.g. the reduction of infectivity of the chitin containing microorganism or pathogen, for example a fungus, insect or nematode, or an overall reduction in the amount of detectable chitin containing microorganism or pathogen, for example a fungus, insect or nematode. “Preventing” refers to absolute prevention, i.e. absence of detectable fungus and/or maintenance of normal levels of detectable chitin containing microorganism or pathogen, for example a fungus, insect or nematode, or reduction or alleviation of the extent of timing (e.g. delaying) of the infection with said chitin containing microorganism or pathogen, for example a fungus, insect or nematode.
An “effective amount” is an amount effective to inhibit the infection, germination or growth of a relevant chitin containing microorganism or pathogen, for example a fungus, insect or nematode, relative to the infection, germination or growth that is seen in the absence of any such treatment.
As the present invention provides the first demonstration of the chitin degradation enhancing properties of non-hydrolytic CBPs, it can be seen that such non-hydrolytic CBPs as described herein are targets in the control of chitin metabolism, for example in chitin containing plague organisms. It is known that several plague organisms (e.g. insects, nematodes and malarial parasites) require chitin degradation during their life cycles. Thus, the present invention further provides a method for combating or treating diseases caused by chitin containing plague organisms such as those described above, which method comprises the administration to an organism of an agent which targets a non hydrolytic CBP as defined herein. Examples of suitable agents include antibodies direct to non hydrolytic CBPs as defined herein, or agents that serve to reduce the expression of non hydrolytic CBPs e.g. at the transcriptional or translational level (e.g. antisense molecules, ribozymes).
As noted above, chitin is present in fungal cell walls and the walls and membranes of other pathogens and parasites, and chitinases have antibiotic action against such organisms. It should therefore be possible to combat plant fungal infections and diseases in plants caused by other chitin containing pathogens, e.g. insects or nematodes by taking advantage of the newly identified properties of non-hydrolytic CBPs to improve, enhance or weaken chitin degradation. Thus, the present invention provides utility in such exemplary applications as enhancing disease resistance in plants, particularly crop plants such as maize.
Plants expressing chitinases have been previously shown to have improved resistance to infection with chitin containing pathogens (see for example, Benhamou, N., K. Broglie, et al. (1993). “Antifungal Effect on Bean Endochitinase on Rhizoctonia-Solani—Ultrastructural-Changes and Cytochemical Aspects of Chitin Breakdown.” Canadian Journal of Microbiology 39(3): 318-328, Bolar, J. P., J. L. Norelli, et al. (2000). “Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor.” PHYTOPATHOLOGY 90(1): 72-77, Herrera-Estrella, A. and I. Chet (1999). Chitinases in biological control Chitin and Chitinases. P. Jollès and R. A. A. Muzzarelli, Birkhäuser: 171-84 and Ding, X., B. Gopalakrishnan, et al. (1998). “Insect resistance of transgenic tobacco expressing an insect chitinase gene.” Transgenic Res 7(2): 77-84.
Plants that have been engineered to express non-hydrolytic CBPs are therefore likely to similarly have improved or probably even better resistance to chitin containing pathogens.
A further embodiment of the invention relates to a transgenic plant comprising an exogenous nucleic acid molecule comprising a sequence encoding a non-hydrolytic CBP as defined herein.
The transgenic plant may further comprise an exogenous nucleic acid molecule comprising a sequence encoding a chitin hydrolytic, e.g. chitinase enzyme, as defined herein.
These plants preferably have improved or enhanced resistance to chitin containing pathogens in that they show reduced rates of infectivity when compared to non-transgenic or wild type plants of the same species (i.e. exposure to higher doses, or quantities or exposure to more virulent chitin containing pathogens are required in order for the plants to become infected with chitin containing pathogens). Alternatively, improved resistance to chitin containing pathogens can be apparent in diminishment of the pathogen when compared to non-transgenic or wildtype plants of the same species.
Without being bound by theory, this may come about by any means, including, but not limited to improved lysis of the pathogen (e.g. fungal pathogen) through weakening of the cell wall.
By “transgenic plant” it is meant a plant which comprises within its genome an exogenous polynucleotide. Generally, the exogenous polynucleotide is integrated, preferably stably integrated within the genome such that the polynucleotide is passed on to successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of exogenous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
By “encoding” it is meant that the nucleic acid comprises the necessary information for translation into the specified protein to be carried out. A nucleic acid molecule encoding a protein may contain additional non-translated sequences (e.g. introns).
Exogenous in reference to a nucleic acid or protein molecule is one that originates from a different species, or if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a microbial sequence is an exogenous sequence when found in a transgenic plant.
Generally the exogenous nucleic acid sequence will be operably linked, i.e. functionally linked to a regulatory sequence.
The term “regulatory sequence” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Preferably, the regulatory elements are ones that operational in plants, for example, a plant promoter and plant polyadenylation recognition sequence.
As used herein, the term “promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate elements of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium and Rhizobium. Examples of promoters under development control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds.
New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
Preferably, the plant is a monocot or dicot. Most preferably, the plant is a crop plant, e.g. Arabidopsis ssp., Euphorbia ssp. particularly Euphorbia pulcherrima, Begonia x cheimantha, Begonia x hiemalis, Begionia x tuberhybrida, Begonia semperflorens, Campanula ssp., Brassica ssp., or Lycopersium esculenium. 
The nucleic acid sequence as referred to herein encodes any non-hydrolytic CBP as defined herein. Most of the appropriate non-hydrolytic CBPs are microbial in origin and it is well known that expression of microbial sequences can be enhanced by modifying the codons that are used such that expression is enhanced in plants. This is particularly useful in the case of expressing a microbial sequence such as the sequence encoding CBP21 in a plant in view of the fact that codon preferences differ in microorganisms and plants. In a preferred embodiment therefore, the nucleic acid sequence contains codons that are preferred for expression in plants (plant high use codons).
Methods for making transgenic plants are well known in the art. The nucleic acid molecule containing the exogenous sequence is typically inserted into a vector e.g. with a regulatory sequence. Typical vectors that are useful for expression of genes in plants are well known in the art and include vectors derived from the tumor induced (Ti) plasmid of Agrobacterium tumefaciens. The non-hydrolytic CBP and the chitin hydrolytic enzyme, e.g. chitnase can be on the same or different vector, under the control of the same or different regulatory sequences.
The vector comprising the sequences from a polynucleotide of the present invention will typically also comprise a marker gene, which confers a selectable phenotype of plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicides basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.
The method of transformation/transfection is not critical to the instant invention; various methods of transformation or transfection are currently available. As newer methods are available to transform crops they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the transformation/transfection may be employed.
Any plant may be transformed. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant cell, i.e. monocot or dicot, target for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., (1986) BioTechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al., (1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment” In Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995); and McCabe et al., (1988) Biotechnology 6:923-926). Also see, Weissinger et al., (1988) Annual Rev. Genet. 22:421-477; Sanford et al., (1987) Particular Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al., (1988) Bio/Technology 6:923-926 (soybean); Datta et al., (1990) Biotechnology 8:736-740 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al., (1988) Biotechnology 6:559-563 (maize); Tomes et al., “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment” in Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995) (maize); Klein et al., (1988) Plant Physiol. 91:440-444 (maize) Fromm et al., (1990) Biotechnology 8:833-839 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London) 311:763-764; Bytebier et al., (1985) In The Experimental Manipulation of Ovule Tissues ed. G. P. Chapman et al., pp. 197-209. Longman, N.Y. (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418; and Kaeppler et al., (1992) Theor. Apply Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
The cells, which have been transformed, may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics is stable maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. One of skill will recognize that after the exogenous nucleic acid molecule is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of number of standard breeding techniques can be used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In see propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plans that would produce the selected phenotype.
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the exogenous nucleic acid molecule. Progeny and variants, and mutant of the regenerated plants are also included within the scope of the invention, provided that these parts compromise the introduced nucleic acid sequences.
A preferred embodiment is a transgenic plant that is homozygous for the added exogenous nucleic acid molecule; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Backcrossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
The particular non-hydrolytic CBP encoding sequence that is introduced can be chosen based on the chitin composition of the pathogen to which it is desired to improve resistance. Approaches for identifying appropriate non-hydrolytic CBPs are discussed elsewhere herein.
Alternatively, the present invention relates to methods of modulating the level of non-hydrolytic CBP in a plant by a) introducing an expression cassette containing a polynucleotide encoding a non-hydrolytic CBP, b) culturing the plant cell under plant cell growing conditions, and c) including expression of the polynucleotide for a time sufficient to modulate the level of non-hydrolytic CBP in the plant.
The method can also be viewed as a method of improving the resistance of a plant to a chitin containing pathogen by a) introducing an expression cassette containing a polynucleotide encoding a non-hydrolytic CBP, b) culturing the plant cell under plant cell growing conditions, and c) inducing expression of the polynucleotide for a time sufficient to modulate the level of non-hydrolytic CBP in the plant.
Pesticide use is now in general criticised as a result of increased environment and health awareness. There is therefore pressure to pursue alternative strategies in pest management. Insect viruses such as baculovirus can also be used a biological pest control systems, in view of the fact that they are able to kill specific insect pests and as such they thus represent as attractive alternative to the use of pesticides as biological pest control systems. These insect viruses are excellent candidates for specifies-specific, narrow spectrum insecticidal applications. Baculoviruses in particular have been shown to have no negative impacts on plants, mammals, birds, fish, or even on non-target insects.
It would be of interest to increase or enhance the infectiousness of insect viruses and one possible way of doing this would be to improve or enhance the ability of such viruses to degrade chitin. The genome of at least some insect viruses contain chitinase genes and genes encoding family 33 non-hydrolytic CBPs (see Table 1) and chitinase activity has been associated with insect virus infectivity. The observation that non-hydrolytic CBPs have a function which is to weaken the structure of chitin and hence to enhance the degradation of chitin thus provides a means for improving the ability of such viruses to weaken or degrade chitin, and a means to enhance the infectivity of such viruses.
The invention thus further provides a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP, wherein said non-hydrolytic CBP is as defined herein.
As used herein “recombinant” includes reference to a virus that has been modified by the introduction of a heterologous or exogenous nucleic acid. Thus, for example, recombinant viruses express genes that are not found in identical form within the native (non-recombinant) form of the virus or express native genes as a result of deliberate human intervention that are otherwise abnormally expressed, under-expressed or not expressed at all. The term “recombinant” as used here does not encompass the alteration of the virus by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
The nucleic acid molecule that encodes the exogenous non-hydrolytic CBP can be placed under the control of appropriate promoters that are known in the art for expression in this biological context. Such promoters may be e.g. inducible or constitutive.
By “exogenous” non-hydrolytic CBP it is meant a non-hydrolytic CBP that is not encoded by the genome of the naturally occurring insect virus, i.e. it originates from a different species, or it has been artificially synthesised. As discussed above, family 33 CBPs such as fusolin are found in insect viruses and the invention excludes naturally occurring viruses.
The nucleic acid molecule that contains the sequence encoding the non-hydrolytic CBP is thus introduced into the insect virus using standard techniques that are well known in the art. The resultant recombinant virus may then also be selected and cultured according to methods that are known in the art.
Alternatively, the invention provides a recombinant insect virus that expresses a naturally occurring non-hydrolytic CBP, under the control of an exogenous promoter, so as to enhance or improve the expression thereof, or to change the expression profile of the protein. By “exogenous” promoter it is meant a promoter that does not normally control the expression of the non-hydrolytic CBP. It may be an insect virus promoter but it is considered to be exogenous if it is not found in its normal context in the recombinant insect virus.
The person skilled in the art can make the appropriate choice of virus for the particular pest or insect, in view of the fact that these viruses are in general host specific.
Similarly, the choice of the non-hydrolytic CBP that is introduced can be make based on the composition of the chitin substrate in the insect that is to be infected.
The invention thus provides a method of increasing the infectivity of an insect virus comprising introducing a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP into the genome of said insect virus.
By improved infectivity it is meant that the insect virus infects its host more efficiently or at lower concentrations of virus than a wild type or unmodified virus does.
In addition to introducing an exogenous nucleic acid sequence encoding a non-hydrolytic CBP into the recombinant virus, it is also possible to introduce a sequence encoding a chitin hydrolytic enzyme such as a chitinase.
The invention further provides a composition comprising a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP together with an appropriate carrier or diluent, or a recombinant insect virus that comprises an non-hydrolytic CBP under the control of an exogenous promoter.
Use of such recombinant insect viruses or compositions in past control is also provided, as in a method of treating a plant or animal comprising administering to said plant or animal an effective amount of a composition comprising a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP together with an appropriate carrier or diluent.
As the present invention provides the first demonstration of the chitin degradation enhancing properties of non-hydrolytic CBPs as defined herein, and assays in which this activity can be assessed and measured, it can be see that the methods and assays of the present invention can be used to identify residues of non-hydrolytic CBPs which are important for the function in enhancing chitin degradation.
Thus, a yet further aspect of the invention provides a method for identifying residues in a non-hydrolytic CBP as defined herein which are important for enabling an enhancement of chitin degradation or for weakening chitin, said method comprising the steps of:                (i) mutating or otherwise altering one or more amino acid residues in the non-hydrolytic CBP, and        (ii) testing the effect of the mutations or alterations on the property of enhancing chitin degradation or weakening chitin.        
In such a method, step (ii) can be performed by assessing the ability of the mutated or otherwise altered non-hydrolytic CBP to enhance the degradation of chitin or weaken chitin, e.g. by using the methods and assays as described elsewhere herein. A comparison of the ability of the mutated or otherwise altered non-hydrolytic CBP to enhance the degradation of chitin or weaken chitin can then be made relative to the non-mutated or otherwise altered non-hydrolytic CBP. The properties of the mutated or otherwise altered non-hydrolytic CBP can thus be compared directly to the wild type, or parent sequence from which mutated or otherwise altered non-hydrolytic CBP is derived.
In such methods, the mutations may have no effect on the enhancement of degradation or may improve or reduce or inhibit the effect on enhancement of degradation. The residues which when mutated have a positive or negative effect on the enhancement of chitin degradation or for weakening chitin are then identified as being important for function. Depending on the proposed use for the non-hydrolytic CBPs such residues are either targets for further mutation (see below) or are identified as residues which should be retained in an unmodified form in order to preserve the important functional property of enhancing chitin degradation.
Alternatively viewed therefore, this provides a method for generating a non-hydrolytic CBP with improved or increased ability to enhance the degradation of chitin or for weakening chitin.
The present invention further provides a method of identifying, developing or producing other non-hydrolytic CBPs which can enhance chitin degradation or can weaken chitin, or variants thereof which have the ability to act on other substrates/polymers, said method comprising the steps of:                (i) obtaining or otherwise providing a non-hydrolytic CBP as defined herein,        (ii) mutating or otherwise altering one or more amino acid residues in the non-hydrolytic CBP,        (iii) assessing the ability of said mutated or altered non-hydrolytic CBP to degrade or to enhance the degradation of the substrate of interest or weaken the substrate of interest,        (iv) identifying mutated or altered non-hydrolytic CBPs which display the desired properties.        
Appropriate substrates for use in the above methods could be any substrate or polymer of interest, in particular any carbohydrate based structure or polymer. Specific and preferred examples include any of the forms of chitin described elsewhere herein (in particular chitin copolymers), cellulose and lignocellulose. As indicated above, such methods can be used to identify, develop or produce binding proteins which weaken a different substrate or enhance the degradation of a different substrate to the starting non-hydrolytic CBP, e.g. a different form of chitin or copolymer or a different substrate such as cellulose or lignocellulose.
The non-hydrolytic CBP used in these methods has the properties as defined elsewhere herein and may be a newly identified non-hydrolytic CBP or may be a known molecule whose properties of enhancement of chitin degradation or weakening of chitin have only become known by use of the methods and assays of the invention as defined elsewhere herein.
If it is the aim of these methods to target a new or different substrate from the one that the starting non-hydrolytic CBP acts on, then preferred residues for mutation or alteration are those which have been identified as being important for binding to chitin and preferably those which have been identified as being important or essential for the enhancement of chitin degradation or weakening of chitin. Preferred residues for mutation are thus those which are exposed at the surface of the binding proteins and in particular those present on the surface which binds to the substrate (the binding surface). In the case of non-hydrolytic CBPs as described herein, preferred residues for mutation in the binding surface are residues which form part of the polar surface patch which is involved in substrate binding (as opposed to a binding surface dominated by aromatic residues such as those found in cellulose binding domains/modules). Preferred residues are thus polar residues (as opposed to aromatic residues) which are capable of forming specific polar interactions, e.g. hydrogen bonds, with the substrate.
In the case of non-hydrolytic CBP21 particularly preferred residues for mutation are at amino acid positions 54, 55, 60, 114, 182 and 185 (i.e. Y54, E55, E60, H114, D182 and N185) which have been shown to be important in the binding of CBP21 to β chitin. Especially preferred residues for mutation are those which have been shown to be important in the ability of CBP21 to enhance chitin degradation or weaken chitin, i.e. at amino acid positions 54, 55, 60, 114 and 182 (i.e. Y54, E55, E60, H114 and D182). In the case of other non-hydrolytic CBPs, preferred residues for mutation are the corresponding residues to those given above for CBP21. Methods for determining the corresponding residues in other non-hydrolytic CBPs are readily available to a person skilled in the art and conveniently will involve sequence alignments using publicly available database as described elsewhere herein and databases such as ClustalX and T-COFFEE, which will allow the identification of corresponding residues. Examples of alignments of other bacterial CBPs with CBP21 and disclosure of corresponding residues and the amino acid position thereof are shown in FIG. 7 (FIG. 2 of Vaaje-Kolstad et al 2005, supra). As more structures of non-hydrolytic CBP-like proteins become available, structural comparisons and structure-based sequence alignments (methods which are readily available to the person skilled in the art), will be of increased utility in the process of identifying mutation targets in the binding surface area. A particularly preferred residue for mutation is the Y54 residue in CBP21 and its corresponding residue in CBP homologues (see for example the corresponding residues shown in FIG. 7). The corresponding residue in CHB1 is Trp57.
Mutation at the residues which are important for the function of enhancing chitin degradation or weakening chitin are likely to result in a modified function, for example an ability to bind to an preferably enhance degradation of or weaken another type of chitin from the original CBP or a different substrate such as those outlined above.
The present invention further provides a method of identifying non hydrolytic CBP homologues or related proteins from other organisms or the same organism, said method comprising the steps of:                (i) obtaining or otherwise providing one or more molecules of interest which are candidate homologue or related protein molecules, and        (ii) testing or screening to assess whether said molecule of interest is a non-hydrolytic CBP which enhances chitin degradation or weakens chitin as defined above, or is a related protein with activities toward other substrates.        
Appropriate substrates for use in the above methods could be any substrate or polymer of interest, in particular any carbohydrate based structure or polymer. Specific and preferred examples include any of the forms of chitin described elsewhere herein (in particular chitin copolymers), cellulose and lignocellulose. As indicated above, such methods can be used to identify homologues or related proteins which enhance the degradation of the same substrate to the known or characterised non-hydrolytic CBP or a different substrate to the known or characterised non-hydrolytic CBP, e.g. a different form of chitin or copolymer or a different substrate such as cellulose or lignocellulose.
The molecules of interest used in step (i) of the method could be single molecules, or could be a library of molecules derived or cloned from one or more species of organism. For example, the molecules could be a library of genes which have been cloned and expressed for testing in accordance with the above method. Any appropriate library of molecules may be used, e.g. cDNA library, genomic fragments, etc. In addition, the library of molecules could represent a library of non-hydrolytic CBP variants, produced e.g. by mutagenesis, which are to be tested for their activities on various substrates.
Although the initial library of molecules may be in the form of nucleic acid sequences, for the test or screening step it is required that the molecules are in the form of proteins (i.e. the nucleic acid molecules need to be expressed). Appropriate methods of expression are well known and described in the art and an appropriate one can be selected. The molecules to be expressed can then be cloned into appropriate expression vectors based on the host cell which is to be used. Preferably for example the molecules to be tested can be cloned into appropriate vectors for expression and preferably secretion from bacterial hosts such as E. coli. Equally however, eukaryotic host cells could be used.
The molecules used in step (i) of the method could be identified or obtained in any appropriate way, e.g. by genome mining, sequence comparison searches, e.g. using sequence databases, etc.
Preferably the bacterial host (or other host) will have some (but preferably a limited) ability to degrade the substrate of interest alone. Optionally the bacterial host can also be engineered to express and secrete an appropriate substrate degrading enzyme, e.g. an appropriate chitinase or cellulase (depending on the substrate of interest). One could then transform these bacteria with a library of protein molecules of interest, e.g. a library of CBP variants, and plate these bacteria on substrate containing plates. An increased halo size would then slow the presence of an effective binding protein.
Alternatively, any appropriate reaction vessel can be used to test the protein molecules. If a library of molecules are to be tested then conveniently each individual protein molecule to be tested is present in an individual reaction vessel, e.g. an assay tube or well. The assays for non-hydrolytic CBPs or related proteins which can enhance the degradation of chitin (or other substrates) can be carried out in such reaction vessels in accordance with the methods of the invention as described herein. For example a certain substrate can be brought into contact with CBPs or related proteins under test (which can either be purified or present in culture supernatants), optionally in combination with an appropriate hydrolytic enzyme, e.g. a chitinase or a cellulase. The degree of substrate degradation can then be monitored, for example by measuring the turbidity in the vessel. Preferred conditions for carrying out these methods to assess substrate degradation are as described elsewhere herein.
In an alternative to the method described above, it would be possible to use a microorganism that can utilize GlcNAc2 as a source of energy, a slow working chitinase and pure crystalline chitin in combination with the various CBPs to be tested. After suitable incubation the mixtures can be analysed for any increase in cell density (turbidity). If the CBP variant is effective, cell growth will be observed. If not, the cells won't grow since the chitinase alone will not produce GlcNAc2 sufficiently rapidly.
Thus, methods of testing or assessing the abilities as described in the above methods can be carried out in accordance with the methods of the invention, in the presence or absence of appropriate chitin hydrolase, e.g. chitinases, as described elsewhere herein. If the methods are designed to identify molecules which are reactive to a non-chitin substrate then appropriate enzymes which facilitate the degradation of these substrates (e.g. appropriate hydrolytic enzymes) can be added in order to assess whether the identified molecule has an effect on enhancing the degradation. For example in the case of cellulose, appropriate cellulases can be added.
In any of the above methods where mutation steps are involved, then any appropriate methods of mutagenesis could be used, for example site directed mutagenesis, random mutagenesis or directed evolution, e.g. random mutagenesis followed by screening. Such techniques are well known to a person skilled in the art.
Substrate binding proteins and proteins which degrade or enhance the degradation of substrate that are identified, developed or produced by any of the above methods and the use thereof to enhance the degradation of substrate form yet further aspects of the invention. These proteins can optionally be manufactured and optionally formulated into compositions for various uses.
In all the above described embodiments, the non-hydrolytic CBPs, and preferred non-hydrolytic CBPs for use in these methods are as defined elsewhere herein.